Application of annonaceous acetogenin in preparation of platinum drug synergist

Abstract

The invention relates to the technical field of biological medicine, in particular to application of annonaceous acetogenin to preparation of a platinum drug synergist. The invention relates to an application of annonaceous acetogenin in preparation of a platinum drug synergist. Experimental data provided by the invention show that the combined action of the annonaceous acetogenin with the concentration of 2.5 mu M and the carboplatin with the concentration of 10 mu M has the optimal inhibition effect on various tumor cells, the annonaceous acetogenin has the capability of enhancing the carboplatin-induced ES2 cell DNA damage, and the addition of the annonaceous acetogenin also has the efficacy of remarkably weakening the carboplatin-induced liver and kidney toxic and side effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to the application of annona in the preparation of platinum drug synergists. Background Art

[0002] Cancer is one of the leading causes of death worldwide, and its occurrence and development involve complex molecular mechanisms. Existing clinically used tumor treatment drugs still face problems such as insufficient efficacy, drug resistance and serious side effects. In recent years, drug combination strategies have shown significant potential in tumor treatment, enhancing the therapeutic effect through synergistic effects and reducing the side effects that may be caused by monotherapy. Combination therapy can act on tumor cells through different mechanisms, reduce the resistance of tumor cells to single drugs, and improve the overall therapeutic efficacy.

[0003] Chemotherapy is one of the important means of tumor treatment, which mainly inhibits the proliferation and spread of tumor cells by using anticancer drugs. Chemotherapy is applicable to many types of tumors, including solid tumors and blood tumors, and can effectively improve the survival rate of patients. However, chemotherapy can easily induce drug resistance in tumor cells, reduce the treatment effect, and even lead to chemotherapy failure; in addition, chemotherapy also has serious side effects on patients, such as nausea, vomiting, hair loss, and reduced immune function, which seriously affect the quality of life of patients. In current clinical treatment, although chemotherapy has a series of shortcomings and challenges, it still occupies an important position in tumor treatment. Therefore, exploring new combined treatment strategies to enhance the efficacy and tolerability of chemotherapy and reduce the toxic side effects of chemotherapy is a major research direction for chemotherapy in the future. Summary of the invention

[0004] The purpose of the present invention is to provide an application of annonacin in the preparation of platinum drug synergists. This patent aims to explore the synergistic effect of annonacin combined with chemotherapeutic drugs, especially platinum chemotherapeutic drugs, on the efficacy of chemotherapeutic drugs and the reduction of toxic side effects.

[0005] This patent application aims to propose a new type of tumor drug combination scheme, combining drugs with complementary effects, in order to achieve better therapeutic effects, provide more effective and safe treatment options for tumor patients, and promote the development of personalized medicine.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of annona in preparing a platinum drug synergist.

[0008] The present invention provides the use of annona in preparing a preparation for reducing the toxic and side effects of platinum drugs.

[0009] Preferably, the platinum drug is cisplatin, carboplatin, nedaplatin, oxaliplatin or lobaplatin.

[0010] Preferably, the mass ratio of annona to platinum drugs is 0.5-100:1-200.

[0011] The invention provides a medicine for treating tumors, comprising annona and platinum drugs.

[0012] Preferably, the tumor is ovarian cancer, liver cancer, lung cancer or colorectal cancer.

[0013] Preferably, the mass ratio of annona to platinum drugs is 0.5-100:1-200.

[0014] Preferably, the platinum drug is cisplatin, carboplatin, nedaplatin, oxaliplatin or lobaplatin.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The annonacin described in the present invention is a natural compound extracted from Annona muricata, and has rich pharmacological activities, such as anti-oxidation, anti-inflammatory, neuroprotection, antibacterial, etc. This patent aims to explore the synergistic effect of annonacin and chemotherapeutic drugs, especially platinum-based chemotherapeutic drugs, on the efficacy of chemotherapeutic drugs and the weakening of toxic side effects. This patent application aims to propose a new type of tumor drug combination scheme, combining drugs with complementary effects, in order to achieve better therapeutic effects, provide more effective and safe treatment options for tumor patients, and promote the development of personalized medicine.

[0017] It can be seen from the experimental data provided by the present invention that the combined action of 2.5 μM annonaine and 10 μM carboplatin has the best inhibitory effect on various types of tumor cells. Annonaine has the ability to enhance the ability of carboplatin-induced DNA damage in ES2 cells, and the addition of annonaine also has the effect of significantly reducing the liver and kidney toxic side effects induced by carboplatin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0019] Figure 1 This is a graph showing the inhibitory effects of Anonacin and carboplatin alone and in combination on different tumor cells.

[0020] Figure 2 The combined efficacy analysis of 2.5 μM Annonacin and different doses of carboplatin on A2780 (A) and ES2 (B).

[0021] Figure 3 The anti-proliferation ability of the combined action of 2.5μM Annonacin and 10μM carboplatin on A2780 and ES2 cells; A: A2780 and ES2 cell colony formation diagram; B: A2780 and ES2 cell colony number diagram.

[0022] Figure 4 The combined effect of 2.5 mg / kg Annonacin and 10 mg / kg carboplatin inhibited the growth of ES2-loaded nude mouse heterotopic transplanted tumors; A: comparison of tumor sizes in different groups; B: comparison of tumor weights in different groups.

[0023] Figure 5 The combined effect of 2.5 μM Annonacin and 10 μM carboplatin enhances the ability of carboplatin to induce DNA damage in ES2 cells; A: fluorescence images of cells in different groups; B: fluorescence expression intensity image.

[0024] Figure 6 The combined effect of 2.5 mg / kgAnnonacin and 10 mg / kg carboplatin significantly reduced the liver and kidney toxicity induced by carboplatin; A: serum alanine aminotransferase (ALT) level; aspartate aminotransferase (AST) level; B: creatinine (Cr) level. DETAILED DESCRIPTION

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Example 1

[0027] 1. Evaluation of in vitro antitumor activity of drug combinations:

[0028] (1) The IC50 (half-maximal inhibitory concentration) of carboplatin and annonacin on tumor cells was calculated using the MTT experiment. The dosing groups were: carboplatin group (a total of 8 concentration gradients were set: 200μM, 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, 1.5625μM), and annonacin group (a total of 8 concentration gradients were set: 100μM, 50μM, 25μM, 12.5μM, 6.25μM, 3.125μM, 1.5625μM, 0.78125μM);

[0029] (2) At the IC50 values ​​of carboplatin and Annonacin, the effects of carboplatin and Annonacin alone and in combination on the viability of tumor cells (ovarian cancer cells A2780 and ES2, liver cancer cells Huh-7, lung cancer cells A549, and colorectal cancer cells HCT116) were evaluated by MTT method to evaluate the anti-tumor activity of the combination drug.

[0030] MTT method to determine cell viability and IC50:

[0031] 1. Place the cells to be tested into a 96-well plate at a density of 5000 cells (dissolved in 160 μL culture medium) per well, overnight to allow the cells to fully adhere to the plate and grow.

[0032] 2. Prepare culture medium containing different concentration gradient drugs and add it to the corresponding culture wells (40 μL per well). Then place the culture plate in a cell culture incubator and continue culturing for 48 hours.

[0033] 3. Add 20 μL MTT solution (5 mg / mL) to each culture well and continue incubation for 4 h.

[0034] 4. Take out the culture plate, carefully discard the supernatant, add 100 μL DMSO to each well, and shake on a shaker for 3 minutes.

[0035] 5. Read the absorbance (OD value) of each well at 570 nm on a microplate reader.

[0036] 6. Calculate the cell survival rate according to the OD value using the following formula: Cell survival rate % = (OD of treatment group / OD of control group) × 100.

[0037] 7. Calculate the IC50 value using nonlinear regression fitting (GraphPad Prism 9).

[0038] Table 1 IC50 (μM) of carboplatin and annonacin against tumor cells determined by MTT method

[0039] Tumor cells Carboplatin Annonacin A2780 75.58±2.69 10.21±0.78 ES2 71.50±7.96 15.12±0.91 Huh-7 32.46±2.06 17.77±2.58 A549 16.32±1.86 21.45±0.87 HCT116 48.25±4.13 19.64±1.03

[0040] from Figure 1 It can be seen that carboplatin and Annonacin have good inhibitory activity against a variety of tumor cells when used separately, and the inhibitory effect is significantly enhanced when carboplatin and Annonacin are used in combination.

[0041] Example 2

[0042] 1. Analysis of drug combination index:

[0043] (1) The cell survival rate (Fu) and cell inhibition rate (Fa) of A2780 and ES2 cells after the drug was applied were obtained by MTT assay. The drug-treated groups were:

[0044] ①5μM Carboplatin+2.5μM Annonacin group;

[0045] ②10μM Carboplatin+2.5μM Annonacin group;

[0046] ③20μM Carboplatin+2.5μM Annonacin group;

[0047] ④40μM Carboplatin+2.5μM Annonacin group;

[0048] ⑤80μM Carboplatin+2.5μM Annonacin group;

[0049] ⑥160μM Carboplatin+2.5μM Annonacin group;

[0050] There were 6 drug-administered groups in total, and the drug action time was 48 h. MTT was then added to measure the absorbance.

[0051] (2) The Chou-Talalay method is used to evaluate the combined effect of drugs, that is, under the condition that the number of drugs and the dosage ratio can be determined, the dose-effect relationship of each drug alone and in combination is quantitatively measured by MTT, and the equivalent dose-effect comparison and the combined effect of drugs are qualitatively and quantitatively described, and it is determined whether a given combination will obtain a synergistic effect (CI<1). By using the "CalcuSyn" software according to the mathematical model established by Chou-Talalay as follows, the Dm, m and CI values ​​are calculated, and a more intuitive Fa-CI diagram of the equivalent dose-effect ratio and the equivalent diagram are drawn.

[0052] Fa / Fu=(D / Dm)m

[0053] log(Fa / Fu)=mlog(D)-mlog(Dm)

[0054] Dx=Dm(Fa / Fu)^1 / m

[0055] CI=(D)1 / (Dx)1+(D)2 / (Dx)2

[0056] Where D: single drug dose; Fa: inhibition rate; Fu: survival rate; Dm: median effective dose; m: reaction kinetic order; CI: drug combination index.

[0057] The strength and nature of drug-drug interactions can be quantitatively determined based on the value of the CI value. When CI>1, the two drugs are antagonistic, when CI=1, the two are additive, and when CI<1, the two are synergistic.

[0058] Results: Chou-Talalay is considered to be one of the simplest and most widely used methods to quantify synergy or antagonism. In order to further explore the drug combination of Annonacin and carboplatin, we fixed the drug dose of Annonacin at 2.5μM, changed the dose of carboplatin (5μM, 10μM, 20μM, 40μM, 80μM, 160μM), and acted on A2780 and ES2 cells for 48 hours. The cell survival rate of each group was obtained by MTT experiment; then the relationship between drug concentration and effect was simulated by CalcuSyn software through the median effect equation model. The results Figure 2 Our experimental results show that the combined use of Anonacin and carboplatin at all doses has a synergistic effect, especially when the dose of carboplatin is 10μM, the CI index is the smallest, indicating that the synergistic effect is the strongest and the drug efficiency Fa is the highest. Therefore, in subsequent experiments, the drug concentration is set to 2.5μM Anonacin + 10μM carboplatin.

[0059] 2. Colony cloning experiment to evaluate the inhibitory effect of combined drug therapy on tumor cell proliferation: Take the cells to be tested in the logarithmic growth phase, digest, centrifuge, and resuspend. Take 10μL of cell suspension, add it to the cell counting plate, and count it in the automatic cell counter. According to the counting results, spread 800 cells per well into a 12-well plate. Place it in a 37℃ constant temperature CO 2 Incubator. After the cells are completely attached, they are treated with culture medium containing 10μM carboplatin, 2.5μM Annonacin, 10μM carboplatin + 2.5μM Annonacin, or an equal volume of culture medium containing DMSO for one week. When the cell density of the DMSO group reaches 80% to 90%, the culture medium is removed, and the cells are gently washed once with PBS, and the PBS is removed. Then the cells are fixed with 4% paraformaldehyde at room temperature for 15 minutes, and washed three times with PBS, 5 minutes each time, to remove the excess fixative.

[0060] Then add 1ml PBS to wash the cells three times, 5min / time, and remove PBS. Add 500μL of 0.04% crystal violet solution to each well, stain for 5min in the dark, and remove the crystal violet. Wash the cells three times with pure water, 5min / time, and remove the pure water. Photograph the results with an optical microscope. After that, use 33% glacial acetic acid to dissolve the crystal violet in 500μL per well. Use Spectra Max M2 multifunctional microplate reader to measure the absorbance of each well at 560nm and record the experimental results. All experiments were repeated three times, and the average of the three experiments was taken and the error was calculated and then plotted using GraphPad 5.0.

[0061] Colony cloning assays are often used to indicate the survival and proliferation ability of individual cells and are therefore well suited for evaluating the anti-tumor activity of drugs in vitro. Figure 3 It can be seen that the number of monoclonal tumor cells in the control group is relatively large, while the number of cell clones is significantly reduced after carboplatin and Anonacin administration, and the number of cell clones is further reduced after carboplatin and Anonacin are co-administered. This result shows that the combined administration of carboplatin and Anonacin has a good synergistic anti-tumor cell proliferation effect.

[0062] 3. ES2 heterotopic transplant tumor experiment to evaluate the in vivo anti-tumor activity: ES2 cells (human ovarian clear cell carcinoma cells) were cultured and expanded to make the cells in the logarithmic growth phase and have strong vitality. Digested and centrifuged, resuspended and washed three times with PBS buffer to avoid the residual serum in the cells stimulating the nude mice and affecting the tumor formation after cell inoculation. The cells were counted with a cell counter to ensure that 5 million cells were implanted in each mouse. The cell suspension was placed in a sterile ice box and quickly inoculated into the right forelimb of BALB / C nude (4 weeks old, female mice, weighing about 20g) mice at 0.2mL / mouse. After the inoculation, the mice were continued to be raised under SPF conditions and the growth of the tumor was observed every other day. The tumor volume was measured with a vernier caliper. When the average volume of the tumor grew to 120mm 3 Drug administration began, and the mice with successful modeling were randomly divided into four groups, each with 6 mice, which were set as solvent control group (0.5% CMC-Na solution), carboplatin group (10 mg / kg), Annonacin group (2.5 mg / kg), and 10 mg / kg carboplatin + 2.5 mg / kg Annonacin group. The above combination drugs and solvent control were administered once a day through tail vein administration, and 7 times were administered midway. The mice died suddenly, the tumors were removed, the mass of the tumors was weighed, and statistical analysis was performed using GraphPad Prism 5.0, and the analysis method was student'test analysis.

[0063] Heterotopic transplant tumor experiments are currently the most commonly used animal models to evaluate the anti-tumor activity of drugs. Therefore, in order to further evaluate the in vivo anti-tumor activity of Annonacin and carboplatin alone and in combination, we constructed an ES2 ovarian cancer cell heterotopic transplant tumor model. Figure 4 It can be seen from the records that compared with the inhibitory effect of Annonacin and carboplatin alone on tumor growth, the combination of the two has a stronger tumor growth inhibitory effect.

[0064] 4. Annonacin enhances the ability of carboplatin to induce DNA damage: Wash the sterilized round coverslips in advance, rinse them with PBS three times, put them in a 12-well plate, and rinse them with the corresponding culture medium once. Digest and centrifuge the cells in the logarithmic growth phase, suspend them, count them, and evenly spread them in a 12-well plate at a density of 30,000 / well, and continue to culture them in an incubator. After the cells adhere to the wall and restore their original morphology, set up groups, 1 blank control group and 3 drug-added groups (10μM carboplatin, or 2.5μM Annonacin, or 10μM carboplatin + 2.5μM Annonacin). After adding the drug, continue to return to the incubator and incubate for 48h. Remove the culture medium and rinse once with PBS. Add 1mL4% paraformaldehyde to each well to fix the cells, fix them on a flat table at room temperature for 15min, and wash them three times with PBS, 5min / time. Add 1mL of permeabilization solution to each well, and permeabilize them on a horizontal shaker at room temperature for 30min to increase the permeability of the cell membrane to antibodies. Discard the permeabilization solution and wash each well with 1mL PBS 3 times, 10min / time. Add 1mL 5% GS to each well to block the cells and block at room temperature for 1h to block nonspecific antibody binding sites. Invert the coverslip with cells onto the corresponding 25μL primary antibody droplet to avoid bubbles. Place in a humidified box and incubate at 4℃ overnight. Add 1mL PBST to each well and wash on a shaker 6 times, 10min each time. Incubate the fluorescent secondary antibody so that the cell face is facing down onto the corresponding 25μL fluorescent secondary antibody droplet to avoid bubbles. Place the slide in a humidified box and incubate at room temperature in the dark for 1.5h. Wash with PBS 3 times, 5min each time. Then dehydrate with 70%, 95%, and 100% ethanol gradients once, on a horizontal table, 5min each time. After dehydration, dry the round slide in the dark. Add 6μL of anti-fading agent containing DAPI to the adhered slide and carefully invert the coverslip on it to avoid bubbles. Seal the slide with nail polish and mark it. Observe and take pictures under an upright fluorescence microscope at 100 times magnification.

[0065] The anti-tumor mechanism of carboplatin is to induce DNA damage, leading to the death of tumor cells. To further explore the enhanced anti-tumor activity of Annonacin combined with carboplatin, we further evaluated its ability to induce DNA damage. Figure 5It can be seen that the combination of Annonacin and carboplatin can further enhance the ability of carboplatin to induce DNA damage, causing more DNA damage in cells.

[0066] 5. Evaluation of combined medication to mitigate the liver and kidney toxicity of carboplatin: The detection of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (Cr) levels is mainly carried out through relevant kits. The specific operations are as follows: ① Determination of ALT and AST content: According to the instructions provided by each kit, first prepare the respective ALT and AST standard curves, take blood from mice at the end of drug treatment through the eyeball, place the blood on a centrifuge at 4000rpm under the following parameters, centrifuge for 15min, take the supernatant and directly measure it. The OD value of each well is measured by an enzyme marker with a wavelength of 510nm. Use the absolute OD value (OD value of the measurement well - OD value of the control well) to check the standard curve and obtain the corresponding ALT / GPT or AST / GPT activity. ② Determination of Cr content: For specific operations, please refer to the instructions, where K is the dilution factor. K = (sample amount + volume of enzyme solution A) / (sample amount + volume of enzyme solution A + volume of enzyme solution B) = 186 / 246; calculation formula: creatinine content (μmol / L) = (ΔA measurement - ΔA blank) / (ΔA standard - ΔA blank) * C standard.

[0067] Carboplatin-induced liver and kidney damage is one of its main clinical side effects. Clinically, the main indicators of liver function tests are transaminases, while the main indicators of kidney function tests are creatinine. In order to evaluate the damage of Annonacin and carboplatin combined with liver and kidney in mice, we detected the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and creatinine (Cr) in serum. Figure 6 It can be seen that the administration of carboplatin significantly increased the levels of ALT / AST and Cr in serum, indicating that it induced liver and kidney damage in mice; Annonacin alone did not affect the levels of ALT / AST and Cr, indicating that it had no effect on the liver and kidney of mice; however, the combination of Annonacin and carboplatin could significantly reduce the increase in ALT / AST and Cr levels induced by carboplatin, indicating that it could reduce the liver and kidney toxicity of carboplatin.

[0068] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of annona in the preparation of platinum drug synergists.

2. Application of annona in the preparation of preparations that reduce the toxic and side effects of platinum drugs.

3. The use according to claim 1 or 2, characterized in that: The platinum drug is cisplatin, carboplatin, nedaplatin, oxaliplatin or lobaplatin.

4. The use according to claim 1 or 2, characterized in that: The mass ratio of annona to platinum drugs is 0.5-100:1-200.

5. A drug for treating tumors, characterized in that: These include annona and platinum drugs.

6. The drug for treating tumors according to claim 5, characterized in that: The tumor is ovarian cancer, liver cancer, lung cancer or colorectal cancer.

7. The drug for treating tumors according to claim 5, characterized in that: The mass ratio of annona to platinum drugs is 0.5-100:1-200.

8. The drug for treating tumors according to claim 5, characterized in that: The platinum drug is cisplatin, carboplatin, nedaplatin, oxaliplatin or lobaplatin.

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